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Iranian Journal of Pharmaceutical Research (2016), 15 (1): 213-220
Received: January 2014
Accepted: Jully 2014
Copyright © 2016 by School of Pharmacy
Shaheed Beheshti University of Medical Sciences and Health Services
Original Article
FTIR Biospectroscopy Investigation on Cisplatin Cytotoxicity in Three
Pairs of Sensitive and Resistant Cell Line
Ensieh Farhadia, Farzad Kobarfardb and Farshad H. Shirazic*
Department of Toxicology and Pharmacology, Shahid Beheshti University of Medical
Sciences, Tehran, Iran. bDepartment of Medicinal Chemistry, Shahid Beheshti University of
Medical Sciences, Tehran, Iran. cPharmaceutical Sciences Research Center, Shahid Beheshti
University of Medical Sciences, Tehran, Iran.
a
Abstract
Fourier Transformed Infrared Spectroscopy (FTIR) has extensively been used for biological
applications. Cisplatin is one the most useful antineoplastic chemotherapy drugs for a variety
of different human cancers. One of the clinical problems in its application, which would
consequently affect the therapeutic outcome of its application, is the occurrence of resistance
to this agent. In this project three different pairs of sensitive and resistant cell lines of human
ovarian A2780 and its resistant pair of A2780-CP, human ovarian OV2008 and its resistant
pair of C13, and finally human lung carcinoma of HTB56 and its resistant pair of HTB56-CP
were grown in the laboratory under the standard procedure. Saline was exposed to control
cells, whereas 1, 5 and 10 µg/mL of cisplatin was exposed to experimental cells, for 1 h. Cells
were then collected and lyophilized from which spectra were taken. According to our results,
we could not trigger a well-recognized cells biomolecular band at 1015 cm-1, being modified
after exposure to cisplatin in all cell lines. On the other hand, there was a clear dose-dependent
increase in protein β-sheet structure related peaks shift in resistant cell lines after exposure
to cisplatin. This would probably indicate an easier protein interaction site for cisplatin in
the resistant cell lines, which would probably inhibit cisplatin from binding to DNA, as the
cytotoxic target. As a conclusion, FTIR biospectroscopy has proven its potency to identify the
interactions, as well as the false engagement cellular sites for cisplatin in sensitive and resistant
cell lines.
Keywords: Cisplatin Resistance; FTIR; A2780; OV2008; HTB56.
Introduction
Cancer is one of the life-threatening diseases,
killing many people every year. Cisplatin is
one of the most effective anticancer drugs for
the treatment of ovarian, breast, head and neck,
and childhood leukemia (1). One of the most
important factors which is limiting cisplatin
* Corresponding author:
E-mail: [email protected]
therapeutic efficacy in cancer treatment is
the development of resistant tumor cells (2).
Whether the resistance to cisplatin is through
the drug uptake, drug degradation in cells or the
development of cellular proteins that detoxifies
this agent is not yet well clear (3). Investigators
are trying to understand the potential responsible
mechanisms for the resistance to cisplatin, with
the hope to overcome this problem and increase
platinum based cancer chemotherapy outcome
(4).
Farhadi E et al. / IJPR (2016), 15 (1): 213-220
methodology of the American Type Culture
Collection (ATCC, Rockville, MD). Cells were
cultured in DMEM/F12 medium (Gibco BRL,
USA) supplied with 10% heat-inactivated fetal
bovine serum in 37 ºC humidified incubator with
5% CO2. All cellular experiments were done in
triplicate. All experiments were performed on
the exponentially growing cells, after a minimum
of three passages from the initial seed of frozen
stock.
Among the different methods for the
investigation on the cellular mechanisms
is biospectroscopy (5). Using different
spectroscopic methods in this technique,
investigators are trying to make correlations
between the cellular spectral alterations with
the biological events. Although the application
of this technique as a creditable methodology
for scientific investigation is not yet well
established, a very promising future is on the
horizon (6). Fourier Transformed Infra-red
(FTIR) spectroscopy is one the most useful
spectroscopic methods used in biology. Previous
publications have tried to correlate some
particular FTIR spectroscopic regions with some
biomolecular structures (7). Scientists have
also tried to derive biological, pathological and
pharmacological applications from the resulted
data (8-10). Scientific challenges are not only
on the interpretation of biological samples
FTIR spectra, but also on the technical aspects
which are used in acquiring a well reproducible,
accurate and precise spectra.
In this study, we have tried to apply FTIR
spectroscopy to investigate three paired human
tumor cell lines, sensitive and resistance to
cisplatin. The idea is to compare the spectral
patterns of sensitive and resistant cell lines to
each other and look for the most likely related
peaks appeared, disappeared and/or altered
in all three pairs of resistant vs. sensitive cell
lines, and/or following the exposure to cisplatin.
We are presenting data, for the first time, to
support the application of FTIR spectroscopy
for pharmacological investigations, as well as
clues to clarify the mechanism of resistance to
cisplatin in these cell lines.
Chemicals
Cisplatin was the clinical formulation of
1 mg/mL in 0.9% saline supplied by Horner
Laboratories
(Montreal,
Canada).
This
formulation was diluted in additional 0.9% saline
to the appropriate concentrations and incubated
for 1 h at 37 °C before use.
Drug Treatment of cells
Cells were grown in 6-well plates separately
and then were exposed to 1, 5, and 10 μg/mL of
cisplatin in serum-free media to prevent cisplatin
denaturation through the protein binding, based
on the published method (12). Control cells were
exposed to the equivalent volumes of sterile
saline. After one-hour incubation of cells with
the above solutions, media was aspirated and
cells were rinsed three times with saline. Cells
were collected by trypsinization gently in 2 mL
of serum-added media to inhibit extra-digestion
by trypsin. Cells were then collected in 15 mL
cone centrifuge vials, washed twice with saline
and then centrifuged (250 g for 10 min) at room
temperature. The cell pellets in 1 mL saline were
then obtained for analysis. The experiments were
performed in triplicate for each concentration of
cisplatin.
Experimental
Freeze-drying protocol
Cells lyophilization was performed using a
Usifroio model SMH-50 instrument. Readily
collected cells were transformed into specific
lyophilization 10 mL vials and the initial freezing
was performed at -40 °C in 2.5 h. Primary
drying procedure was then conducted on these
cells during 4.5 h at the same temperature at
5x10-2 millibar pressure. Temperature was then
increased gradually up to the room temperature
of 25 °C in 22 h under the same vacuum
Cell lines
Human ovarian carcinoma cell lines of
OV2008 and A2780 (12), and their respected
cisplatin resistant variants of Ov2008-CP (C13)
and A2780CP, Human lung adenocarcinoma
HTB56 (ATCC® HTB-56™) and its cisplatin
resistant variant of HTB56-CP were obtained
from the Pharmacology Lab, Ottawa Regional
Cancer Centre, Ottawa, Ont., Canada. The
cells were cultured following the standard
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FTIR on three pairs cell line
Figure 1. a) The general pattern of FTIR spectra of under study cell lines (refer to text for the name and specification of cell lines), and b)
The FTIR spectra of Human ovarian adenocarcinomas of OV2008 (upper graphs) and its cisplatin resistant variant of A2780-CP (lower
graphs) after exposure to 0, 1, 5 and 10 µg/mL of cisplatin for 1 h, in the region of 600-4000 cm-1.
condition. Vial lead was then sealed using 7x10-3
millibar pressure. These vials containing freezedried cells were kept in refrigerator before the
FTIR experiments.
by Dunnett᾽s post hoc test was used to assess
significant differences (p < 0.05) between spectra
resulted from different cells exposed to different
concentrations of cisplatin. All the statistical
analysis was performed using GraphPad PRISM®
version 5 Software (GraphPad Software, San
Diego, CA, USA).
FTIR analysis
At the time of spectroscopy, vials containing
freeze-dried cells were opened and 5 mg of cell
powder were mixed with 95 mg of potassium
bromide rapidly. A small amount of this mixed
powder was put in the sample holder of a Perkin
Elmer instrument model Spectrum one provided
with a TGF detector, and analyzed using the
DRIFT (Diffuse Reflectance Infra-red Fourier
Transform) method. Pure potassium bromide
was used as the control for background spectra.
For each spectrum, a total of 512 scans, at 4
-1
cm resolution, were applied in the range of 6004000 cm-1. Spectral mathematical manipulations
(as the baseline corrections, smoothing, and
deconvolution) and statistical parameters (such
as peak frequency shifts and intensity ratios of
various infrared bands) were calculated using
Perkin Elmer software 1B version 3.02.
Results
Figure 1 represents the total spectra of human
ovarian adenocarcinoma A2780 and its resistant
variant after 1 h exposure to 1, 5 and 10 µg/mL
of cisplatin, or saline as control, for 1 h in the
range of 600-4000 cm-1. As is shown in these
spectra, there are several features related to the
different cell components that are altered after
the exposure of cells to cisplatin. The area of
above 2000 cm-1 has mainly been correlated with
the CH2 of lipid bi-layer matrix of the cells. Most
of the information on the cellular proteins are
coming from the amide I and II bands represented
in the area of 100-1700 cm-1. Different features
related to DNA and its backbone is absorbed
around the area of 1200 cm-1, followed by
the spectra of cellular carbohydrates in the
neighboring region. Changes, if any, observed in
any particular regions are discussed ahead.
To better understand the cellular spectral
changes in different cell lines, mathematical
revisions are needed. Deconvolution is a wellknown technique to extrapolate different
co-covered peaks from each other. Figure
2 represents a sample of mathematical
deconvolution on A2780 cell line spectra to
Statistical analysis
Different spectral parameters were used for
the comparison purposes between the control
and cisplatin exposed cells, including wave
numbers (cm-1) and absorbance ratios based
on the peak height and area under the curves.
Results, expressed as mean ± S.D in tables and
graphs were created using nonlinear regression
analysis in GraphPad PRISM® Software. Oneway analysis of variance (ANOVA) followed
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Farhadi E et al. / IJPR (2016), 15 (1): 213-220
Figure 2. Example of FTIR spectral deconvulotion on human cell lines representing the result on human ovarian adenocarcinoma of
OV2008 as one example. Deconvulotion helps to identify the peaks with a higher precision.
further clarify some regions of spectra for a better
peak determination and comparison purposes.
Figures 3 and 4 are representing the
alterations of spectra in the ovarian cell lines
sensitive (OV2008) and sensitive (C13) to
cisplatin following exposure to this drug. As are
shown in these figures and are more elaborated
in the discussion section the most changes are
observed around 1200-1700 cm-1 which are more
correlated with the DNA and proteins. Figures
5 and 6 are representing the spectral alterations
in the sensitive and resistance pair of lung cells
alone (concentrations 0) or after exposure to
the different concentrations of cisplatin. Again,
most of the variations are seen in the same area
of 1200-1700 cm-1 coming from cellular DNA
and proteins. As are shown in these figures,
most of the intracellular variations as well as the
sensitivity to cisplatin parameters in these cell
lines should be found among the cellular DNA
or proteins. Not all of these regions and their
alterations have yet been well defined for the
cells; however, those changes which are better
understood based on the previous publications
are elaborated and the biological meanings of
these changes are discussed in the following
section.
Discussion
Cancer chemotherapy success is to a high
extent related to the cellular response to the
selected chemotherapeutic agent. Time has
a major role in the chemotherapy outcome,
although it may take months before the
physician can estimate the tumor response to
the selected medication. A fast and reliable
technique to understand the sensitivity or
resistance of a tumor type to different cancer
chemotherapy agents will save the critical time
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FTIR on three pairs cell line
Figure 3. Comparison of spectral peaks shifts on four peaks corresponding to the cellular absorbance of carbohydrates, symmetrical
and unsymmetrical stretching of phosphate bands in three human cell lines (upper graphs) and their cisplatin resistant variants (CPs in
lower graph).
and will benefit patients. Fourier Transform
Infrared Spectroscopy (FTIR) is one of the most
promising techniques to distinguish biological
variations among different samples. Simplicity,
prompt response, sensitivity and subject
independence properties hallmark this technique
among other conventional diagnostic methods.
Although it is in the beginning and discovering
steps, its advantages on cancer detection have
widely been examined (13) and new applications
are coming by the time.
We have aimed to try this technique for the
estimation of cancer cells sensitivity to cisplatin
cytotoxicity as a model for its application
in chemotherapy outcome prediction use.
Cisplatin is one of the most widely used cancer
chemotherapeutic agents in clinics, and many
sensitive or resistance cell lines to this agent
are recognized and well established. Three
pairs of the same origin, but cisplatin sensitive
and resistant variant cell lines, were selected in
this study and exposed to the clinically relevant
doses of cisplatin for a double period of clinically
relevant half life of this drug. Cells were then
collected, lyophilized and examined under FTIR
instrument to look for a well discriminative peak
to be consistent with sensitivity or resistance
of the cell line to cisplatin. Lyophilization has
been introduced to the collected cells to set up
a repeatable unique method of cell preparation
and long term preservation to be useful for
biospectroscopy, which will also eliminate the
large proportion of cellular and intracellular water
with its related large interfering peak which mask
many spectroscopic peaks from various cellular
biomolecules in FTIR spectroscopy. A good and
acceptable reproducibility in cell spectra have
also been acquired using the lyophilized cells
instead of other sample drying or H2O/D2O
substitute techniques (data not shown).
Figure 4. Comparison of spectral peaks shifts on four peaks corresponding to the cellular absorbance of nucleic acids and DNA bands in
three human cell lines (upper graphs) and their cisplatin resistant variants (CPs in lower graph).
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Farhadi E et al. / IJPR (2016), 15 (1): 213-220
Figure 5. Comparison of spectral peaks shifts on four peaks corresponding to the cellular absorbance of amide bands arising from
proteins in three human cell lines (upper graphs) and their cisplatin resistant variants (CPs in lower graph).
Zendehdel et al (14) have also used FTIR
biospectroscopy to distinguish between one
human sensitive to cisplatin ovarian cell line
with two resistant variant cell lines using PCA
analysis. They have looked at bands in four
different segments of spectra in the ranges of
1000-1500, 1500-2000, 2000-2500 and 25003000 cm-1. Although no discriminative peaks
have been found in the two segments of 15002000 and 2500-3000, bands at 1240, 1330, 1380,
and 1407 cm-1 were clearly recognizing sensitive
from resistant cell lines. As is shown in Figure 5,
a peak at 1645 cm-1 arising from protein β-sheets
is clearly decreased after the exposure of
resistance cell line of HTB-56 after the exposure
to cisplatin indicating the cisplatin interaction
with a protein which affects its conformational
orientation.
The significance of the present work in
comparison to the previous ones is that three
pairs of well-established and confirmed cisplatin
resistant and sensitive cell lines are examined
using FTIR spectroscopy with and without
exposure to this drug. Using such a unique
protocol, we were hoping on not only to test and
prove the application of FTIR biospectroscopy
for these purposes in more detail, but also
to collect hint for the cisplatin sensitivity/
resistance determinants of cellular biomolecular
sites with the study of peaks shifts after exposure
to the different concentrations of cisplatin in
comparison to control.
Among the different tested regions, peaks
at 1015 cm-1 have disappeared or shifted to a
lower wave number after exposure to cisplatin
in almost all resistance cell lines compared to
their matched sensitive variants (Figure 6).
Although this FTIR band has been correlated to
the Cβ-(CH3)2 symmetric stretching in aliphatic
compounds, it has not yet been well recognized
in biological structures of cell lines (15). In any
case, lower wave numbers correlate to higher
frequencies and more required energy for the
vibration. Observed shift of this peak to a lower
wave number after the cell exposure to cisplatin
in our experiments might be related to the
attachment of the heavier and covalently bound
cisplatin to the corresponding group of the
respected biomolecules in these cells as one of
the major cisplatin candidate sites, which need
to be identified in future.
The human ovarian adenocarcinoma
A2780 is also presenting two unique peaks
appearances for which none of other cell lines
are incorporating. The peak at 1080 cm-1 in
cisplatin sensitive A2780 cell line is shown at
1100 in other cell lines, and the peak at 1628
cm-1 in cisplatin resistance A2780-CP cell line is
presented at 1635 in other cell lines. The peak
around 1080 to 1100 cm-1 is correlated to the
symmetric stretching of PO2 groups in cellular
phospholipids. Presence of this peak in a lower
wave numbers in A2780 cell line compared to
the other cancerous cells studied in this project
might indicate a tighter phospholipid structure
of the cell membrane in these cells. This unique
structural feature for A2780 cells has been
mentioned in previous publications of Taylor et
al. when they were investigating the resistance
mechanisms of this cell line to cisplatin (16),
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FTIR on three pairs cell line
Figure 6. Comparison of spectral peaks shifts on four peaks corresponding to the cellular absorbance of CH and CH2 bands arising from
cell bilayer lipid membrane matrix vibration of three human cell lines (upper graphs) and their cisplatin resistant variants (CPs in lower
graph).
analyzing the cell membrane constituent of
ovarian cell lines. The peak around 1625 to 1535
cm-1, on the other hand, has been correlated to
the total cellular protein β-sheet structures (17).
The fact that this peak is presented in a lower
wave number in the resistant variant of A2780
cells means a higher energy (corresponding
to higher frequency) is needed to vibrate these
cellular biomolecules in this cell line. This
phenomenon might be interpreted as cisplatin
covalent binding to the β-sheet structure proteins
instead of the α-structured DNA. Such an event
might well preserve these cells from the cisplatin
active site in other cell lines, which is shown to
be the covalent bind to DNA, introducing a new
hypothesis for the mechanism of resistance to
cisplatin in this cell line.
and a not yet known biomolecule indicated at
1015 cm-1, instead of cisplatin target site on the
α-helical structure of DNA. More investigations
are ongoing to further explore this finding.
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As a conclusion, our work has further
proved the application of FTIR spectroscopy
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to the different concentrations of this agent has
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This article is available online at http://www.ijpr.ir
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